DESCRIPTION: This is a revised proposal to characterize the thrombospondin 4 molecule and determine its function in vivo as well as its evolutionary development as a member of the thrombospondin gene family. The Aims of the original proposal have been maintained and one additional Aim included based on recent advances in the applicant's laboratory. As described in the original proposal, the investigator will clone and sequence human thrombospondin-4 (TS-4) before initiating his studies. Since the time of the original proposal he has isolated several clones of human TS-4 and has almost completed the sequence,which appears to be almost identical to that of Xenopus laevis. The clones for both human and Xenopus TS-4 will be used to prepare fusion proteins. The proteins will be used to induce polyclonal antisera for immunocytochemical studies in tissue. The locations of the TS-4 genes on the human and mouse chromosomes will be determined and the promoter regions characterized. TS-4 will be purified either from culture medium (if secreted) or from cell membranes or cytoplasm and NH2 - terminal sequencing will be performed by a collaborator. The recombinant protein will be expressed into NIH 3T3 cells. The recombinant TS-4 will be characterized with respect to appearance by electron microscopy, interaction with calcium, heparin, and cell-binding properties. It is now known for example, that human TS-4 has an RGD sequence. The new Aim is to delete TS-4 gene from the mouse genome by performing homologous recombination in embryonic stem cells. This will provide chimeric mice from which mice that are homozygous for TS-4 deletion can be bred.This mouse model should provide insight into the biologic significance of TS-4.